MicroRNA Expression in Human Peripheral Blood Microvesicles and Uses Thereof

ABSTRACT

The present invention provides novel methods and compositions for the diagnosis, prognosis and treatment of disorders by examining samples containing microvesicles and miRs therein.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Patent Application 60/993,809 filed Sep. 14, 2007, and 61/055,178 filed May 22, 2008, which are fully incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was not made with any government and the government has no rights in this invention.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs or miRs) are small non-coding RNAs expressed in animals and plants. They regulate cellular function, cell survival, cell activation and cell differentiation during development.^(7;8)

MicroRNAs are a small non-coding family of 19-25 nucleotide RNAs that regulate gene expression by targeting messenger RNAs (mRNA) in a sequence specific manner, inducing translational repression or mRNA degradation depending on the degree of complementarity between miRNAs and their targets (Bartel, D. P. (2004) Cell 116, 281-297; Ambros, V. (2004) Nature 431, 350-355). Many miRs are conserved in sequence between distantly related organisms, suggesting that these molecules participate in essential processes. Indeed, miRs are involved in the regulation of gene expression during development (Xu, P., et al. (2003) Curr. Biol. 13, 790-795), cell proliferation (Xu, P., et al. (2003) Curr. Biol. 13, 790-795), apoptosis (Cheng, A. M., et al. (2005) Nucl. Acids Res. 33, 1290-1297), glucose metabolism (Poy, M. N., et al. (2004) Nature 432, 226-230), stress resistance (Dresios, J., et al. (2005) Proc. Natl. Acad. Sci. USA 102, 1865-1870) and cancer (Calin, G. A, et al. (2002) Proc. Natl. Acad. Sci. USA 99, 1554-15529; Calin, G. A., et al. (2004) Proc. Natl. Acad. Sci. USA 101, 11755-11760; He, L., et al. (2005) Nature 435, 828-833; and Lu, J., et al. (2005) Nature 435:834-838).

There is also strong evidence that miRs play a role in mammalian hematopoiesis. In mice, miR-181, miR-223 and miR-142 are differentially expressed in hematopoietic tissues, and their expression is regulated during hematopoiesis and lineage commitment (Chen, C. Z., et al. (2004) Science 303, 83-86). The ectopic expression of miR-181 in murine hematopoietic progenitor cells led to proliferation in the B-cell compartment (Chen, C. Z., et al. (2004) Science 303, 83-86). Systematic miR gene profiling in cells of the murine hematopoietic system revealed different miR expression patterns in the hematopoietic system compared with neuronal tissues, and identified individual miR expression changes that occur during cell differentiation (Monticelli, S., et al. (2005) Genome Biology 6, R71). A recent study has identified down-modulation of miR-221 and miR-222 in human erythropoietic cultures of CD34⁺ cord blood progenitor cells (Felli, N., et al. (2005) Proc. Natl. Acad. Sci. USA. 102, 18081-18086). These miRs were found to target the oncogene c-Kit. Further functional studies indicated that the decline of these two miRs in erythropoietic cultures unblocks Kit protein production at the translational level leading to expansion of early erythroid cells (Felli, N., et al. (2005) Proc. Natl. Acad. Sci. USA. 102, 18081-18086). In line with the hypothesis of miRs regulating cell differentiation, miR-223 was found to be a key member of a regulatory circuit involving C/EBPa and NFI-A, which controls granulocytic differentiation in all-trans retinoic acid-treated acute promyelocytic leukemic cell lines (Fazi, F., et al. (2005) Cell 123, 819-831).

A frequent deletion and reduced expression of two miRs in B-cell chronic lymphocytic leukemia has been identified. This discovery stimulated numerous articles documenting aberrant expression of miRs in head and neck carcinomas, small cell lung cancers, glioblastomas, breast cancers, chronic lymphocytic leukemia, and Burkitt lymphoma.⁹⁻¹² More recently, a relationship between inflammation and miRs has been reported in macrophages.¹³

In order to test for such disorders, tissue samples have been obtained in order to confirm the presence of such macrophages. In addition, until now, there has been no report demonstrating that microvesicles that circulate in the blood contain miRs.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and advantages of the invention may be realized and attained as particularly pointed out in the appended claims.

SUMMARY OF THE INVENTION

In one aspect, there is provided a method for identifying specific miRs that are present in microvesicles and/or have altered expression levels of specific miRs in tissue, fluids and/or cells.

Microvesicles facilitate communication between cells. Many cells including macrophages, platelets, T-cells, and tumors release small microvesicles containing nucleic acids and/or proteins¹⁻⁵. Factors contained within the microvesicles regulate angiogenesis, cell growth, and cell differentiation^(1;3).

In another aspect, the presence of miRs in such fluids as peripheral blood of patients suffering from particular disorders is determined.

In another aspect, the presence of miRs in lung tissue of patients suffering from pulmonary fibrosis is determined.

In yet another aspect, there is provided herein a method of diagnosing or prognosticating a particular disorder in a subject (e.g., a human). According to one particular method, the level of at least one miR gene product in a test sample from the subject is compared to the level of a corresponding miR gene product in a control sample. An alteration (e.g., an increase, a decrease) in the level of the miR gene product in the test sample, relative to the level of a corresponding miR gene product in the control sample, is indicative of the subject either having, or being at risk for developing, an acute inflammatory disorder.

In one embodiment, the level of the miR gene product in the test sample from the subject is greater than that of the control. In another embodiment, the at least one miR gene product is selected from the group consisting of the miRNAs as shown herein.

In particular embodiments, the disorder that is diagnosed or prognosticated is one that causes mononuclear phagocytes and/or THP-1 cells to release microvesicles.

In particular embodiments, the disorder that is diagnosed or prognosticated is one that causes an inflammatory response.

In another embodiment, the invention is a method of treating a cancer and/or an inflammatory disorder in a subject (e.g., a human).

In one particular method, an effective amount of a compound for inhibiting expression of at least one miR gene product selected from the one or more of the groups found in Table I-VI is administered to the subject.

In one embodiment, the compound for inhibiting expression of at least one miR gene product inhibits expression of a miR gene product selected from one or more of the groups found in Tables I-VI.

The invention further provides pharmaceutical compositions for treating cancer and/or an inflammatory disorder. In one embodiment, the pharmaceutical compositions of the invention comprise at least one miR expression-inhibition compound and a pharmaceutically-acceptable carrier. In a particular embodiment, the at least one miR expression-inhibition compound is specific for a miR gene product whose expression is greater in blood from diseased patients compared to normals.

In yet another embodiment, the pharmaceutical composition further comprises at least one anti-inflammatory agent.

In one embodiment, the invention is a pharmaceutical composition for treating a cancer associated with overexpression of a miR gene product and/or a lung disorder associated with overexpression of a miR gene product. Such pharmaceutical compositions comprise an effective amount of at least one miR gene product and a pharmaceutically-acceptable carrier, wherein the at least one miR gene product binds to, and decreases expression of, the miR gene product. In another embodiment, the at least one miR gene product comprises a nucleotide sequence that is complementary to a nucleotide sequence in the miR-gene product. In still another embodiment, the at least one miR gene product is miR- or a variant or biologically-active fragment thereof. In yet another embodiment, the pharmaceutical composition further comprises at least one anti-cancer agent.

Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the differentiation induced release of microvesicles from macrophages. Peripheral blood monocytes (PBM) were untreated (light) or treated with GM-CSF (dark) for 24 h. Cell-free supernatant was collected and ultracentrifuged. The vesicles were resuspended in PBS and analyzed for size on a flow cytometry. Prior to analysis, FSS and SSC parameters were adjusted using 2 μm standard beads (not shown). Shown is representative data from three different donors.

FIGS. 2A-2C show microvesicles mediate macrophage differentiation. Microvesicles were collected from PMA-treated THP1 cells then added to undifferentiated THP1 cells (FIG. 2B) or monocytes (FIG. 2C). As a control, THP1 cells were left untreated (FIG. 2A). The cells were photographed daily. Shown are the cells at day 3.

FIGS. 3A-3C show the isolation of peripheral blood microvesicles. Following informed consent, plasma was obtained from 20 cc of blood from normal volunteer donors. The microvesicles from 0.5 cc of plasma were incubated with CD206-FITC or MHCII-FITC antibodies and analyzed on BD FACS Calibur for size using forward vs. side scatter (FIG. 3A) and surface antigen expression (FIG. 3B). The percent expression of either CD206 or MHC II compared to isotype control was determined for the gated region shown in FIG. 3A (FIG. 3C). Shown is the average±SEM of two donors.

FIG. 4. Analysis of the origin of peripheral blood microvesicles. Peripheral blood microvesicles from healthy donors (n=10) were analyzed by flow cytometry. To determine cell origin, microvesicles were stained for CD3, CD202b (Tie-2), CD66b, CD79a, or CD41a to determine those that originated from T-cells, endothelial cells, neutrophils, B-cells, or platelets. Mononuclear phagocyte-derived microvesicles were positive for CD14, CD206, CCR3, CCR2, or CCR5. Shown is the average % maximum of total gated events±S.E.M.

FIGS. 5A-5D. miRNA expression from peripheral blood microvesicles and PBMC. (FIG. 5A) Hierarchal cluster analysis for microvesicles and PBMC is shown based on filtering criteria. Heat-maps demonstrating the expression profile for microvesicles (FIG. 5B) and PBMC (FIG. 5C) were generated. (FIG. 5D). The number of shared and specific for each sample group is shown.

FIG. 6: Table I showing various diseases and up- and down-regulated miRs associated therewith. microRNAs that are important in tissue of human diseases, including cancer and non-cancer applications are listed. Comparing miRNAs that are undetectable in the plasma from our data set (FIG. 7, Table II) with miRNAs known to increase in the tissue of specific diseases, the inventors now believe that we predict that several miRNAs may serve as biomarkers in the plasma (see miRs in bold in FIG. 6, Table I Increase Expression Column).

FIG. 7: Table II showing miRs that are expressed in the plasma and those that are undetectable.

FIG. 8: Table III lists miRs and show the top ten expressed miRNAs in the plasma microvesicles and the PBMC from all individuals.

FIG. 9: Table IV showing canonical pathways involved in metabolism and regulation of the acquired immune system were highly regulated by the expression of these miRNAs using Sanger miRBase alone (top) or common targets from Sanger miRBase and TargetScan (bottom).

FIG. 10: Table V showing that 20 miRNAs had more than a three-fold increase in expression in the PBMC fraction compared to the microvesicles plasma samples as well as the fold change in plasma microvesicles compared to PMBC (last Column)

FIG. 11: Table VI showing, the normalized expression data for all detected miRs: detector name, ave-MNC, std-MNC, detector name, ave-serum, std-serum.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is based, in part, on the identification of specific microRNAs (miRNAs) that are involved in an inflammatory response and/or have altered expression levels in blood. The invention is further based, in part, on association of these miRNAs with particular diagnostic, prognostic and therapeutic features.

As described and exemplified herein particular miRNA are up- or down-regulated during tissue injury and/or inflammation.

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,” “miR” or “miRNA” refers to the unprocessed or processed RNA transcript from a miR gene. As the miR gene products are not translated into protein, the term “miR gene products” does not include proteins. The unprocessed miR gene transcript is also called a “miR precursor,” and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miR precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, RNAse III (e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the “processed” miR gene transcript or “mature” miRNA.

The active 19-25 nucleotide RNA molecule can be obtained from the miR precursor through natural processing routes (e.g., using intact cells or cell lysates) or by synthetic processing routes (e.g., using isolated processing enzymes, such as isolated Dicer, Argonaut, or RNAse III). It is understood that the active 19-25 nucleotide RNA molecule can also be produced directly by biological or chemical synthesis, without having to be processed from the miR precursor. When a microRNA is referred to herein by name, the name corresponds to both the precursor and mature forms, unless otherwise indicated.

The present invention encompasses methods of diagnosing or prognosticating whether a subject has, or is at risk for developing, a disorder where microvesicles are released.

The methods comprise determining the level of at least one miR gene product in a sample from the subject and comparing the level of the miR gene product in the sample to a control. As used herein, a “subject” can be any mammal that has, or is suspected of having, such disorder. In a preferred embodiment, the subject is a human who has, or is suspected of having, such disorder.

The level of at least one miR gene product can be measured in cells of a biological sample obtained from the subject.

In another embodiment, a sample can be removed from the subject, and DNA can be extracted and isolated by standard techniques. For example, in certain embodiments, the sample can be obtained from the subject prior to initiation of radiotherapy, chemotherapy or other therapeutic treatment. A corresponding control sample, or a control reference sample (e.g., obtained from a population of control samples), can be obtained from unaffected samples of the subject, from a normal human individual or population of normal individuals, or from cultured cells corresponding to the majority of cells in the subject's sample. The control sample can then be processed along with the sample from the subject, so that the levels of miR gene product produced from a given miR gene in cells from the subject's sample can be compared to the corresponding miR gene product levels from cells of the control sample. Alternatively, a reference sample can be obtained and processed separately (e.g., at a different time) from the test sample and the level of a miR gene product produced from a given miR gene in cells from the test sample can be compared to the corresponding miR gene product level from the reference sample.

In one embodiment, the level of the at least one miR gene product in the test sample is greater than the level of the corresponding miR gene product in the control sample (i.e., expression of the miR gene product is “upregulated”). As used herein, expression of a miR gene product is “upregulated” when the amount of miR gene product in a sample from a subject is greater than the amount of the same gene product in a control (for example, a reference standard, a control cell sample, a control tissue sample).

In another embodiment, the level of the at least one miR gene product in the test sample is less than the level of the corresponding miR gene product in the control sample (i.e., expression of the miR gene product is “downregulated”). As used herein, expression of a miR gene is “downregulated” when the amount of miR gene product produced from that gene in a sample from a subject is less than the amount produced from the same gene in a control sample. The relative miR gene expression in the control and normal samples can be determined with respect to one or more RNA expression standards. The standards can comprise, for example, a zero miR gene expression level, the miR gene expression level in a standard cell line, the miR gene expression level in unaffected samples of the subject, or the average level of miR gene expression previously obtained for a population of normal human controls (e.g., a control reference standard).

The level of the at least one miR gene product can be measured using a variety of techniques that are well known to those of skill in the art (e.g., quantitative or semi-quantitative RT-PCR, Northern blot analysis, solution hybridization detection). In a particular embodiment, the level of at least one miR gene product is measured by reverse transcribing RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to one or more miRNA-specific probe oligonucleotides (e.g., a microarray that comprises miRNA-specific probe oligonucleotides) to provide a hybridization profile for the test sample, and comparing the test sample hybridization profile to a hybridization profile generated from a control sample. An alteration in the signal of at least one miRNA in the test sample relative to the control sample is indicative of the subject either having, or being at risk for a particular disorder.

Also, a microarray can be prepared from gene-specific oligonucleotide probes generated from known miRNA sequences. The array may contain two different oligonucleotide probes for each miRNA, one containing the active, mature sequence and the other being specific for the precursor of the miRNA. The array may also contain controls, such as one or more mouse sequences differing from human orthologs by only a few bases, which can serve as controls for hybridization stringency conditions. tRNAs and other RNAs (e.g., rRNAs, mRNAs) from both species may also be printed on the microchip, providing an internal, relatively stable, positive control for specific hybridization. One or more appropriate controls for non-specific hybridization may also be included on the microchip. For this purpose, sequences are selected based upon the absence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. For example, probe oligonucleotides of an appropriate length, e.g., 40 nucleotides, are 5′-amine modified at position C6 and printed using commercially available microarray systems, e.g., the GeneMachine OmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides. Labeled cDNA oligomer corresponding to the target RNAs is prepared by reverse transcribing the target RNA with labeled primer. Following first strand synthesis, the RNA/DNA hybrids are denatured to degrade the RNA templates. The labeled target cDNAs thus prepared are then hybridized to the microarray chip under hybridizing conditions, e.g., 6×SSPE/30% formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT at 37° C. for 40 minutes. At positions on the array where the immobilized probe DNA recognizes a complementary target cDNA in the sample, hybridization occurs. The labeled target cDNA marks the exact position on the array where binding occurs, allowing automatic detection and quantification. The output consists of a list of hybridization events, indicating the relative abundance of specific cDNA sequences, and therefore the relative abundance of the corresponding complementary miRs, in the patient sample. According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeled primer. The microarray is then processed by direct detection of the biotin-containing transcripts using, e.g., Streptavidin-Alexa647 conjugate, and scanned utilizing conventional scanning methods. Image intensities of each spot on the array are proportional to the abundance of the corresponding miR in the patient sample.

The use of the array has several advantages for miRNA expression detection. First, the global expression of several hundred genes can be identified in the same sample at one time point. Second, through careful design of the oligonucleotide probes, expression of both mature and precursor molecules can be identified. Third, in comparison with Northern blot analysis, the chip requires a small amount of RNA, and provides reproducible results using 2.5 μg of total RNA. The relatively limited number of miRNAs (a few hundred per species) allows the construction of a common microarray for several species, with distinct oligonucleotide probes for each. Such a tool allows for analysis of trans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specific miRs, a microchip containing miRNA-specific probe oligonucleotides corresponding to a substantial portion of the miRNome, preferably the entire miRNome, may be employed to carry out miR gene expression profiling, for analysis of miR expression patterns. Distinct miR signatures can be associated with established disease markers, or directly with a disease state.

According to the expression profiling methods described herein, total RNA from a sample from a subject suspected of having a particular disorder is quantitatively reverse transcribed to provide a set of labeled target oligodeoxynucleotides complementary to the RNA in the sample. The target oligodeoxynucleotides are then hybridized to a microarray comprising miRNA-specific probe oligonucleotides to provide a hybridization profile for the sample. The result is a hybridization profile for the sample representing the expression pattern of miRNA in the sample. The hybridization profile comprises the signal from the binding of the target oligodeoxynucleotides from the sample to the miRNA-specific probe oligonucleotides in the microarray. The profile may be recorded as the presence or absence of binding (signal vs. zero signal). More preferably, the profile recorded includes the intensity of the signal from each hybridization. The profile is compared to the hybridization profile generated from a normal control sample or reference sample. An alteration in the signal is indicative of the presence of, or propensity to develop, the particular disorder in the subject.

Other techniques for measuring miR gene expression are also within the skill in the art, and include various techniques for measuring rates of RNA transcription and degradation.

The invention also provides methods of diagnosing whether a subject has, or is at risk for developing, a particular disorder with an adverse prognosis. In this method, the level of at least one miR gene product, which is associated with an adverse prognosis in a particular disorder, is measured by reverse transcribing RNA from a test sample obtained from the subject to provide a set of target oligodeoxynucleotides. The target oligodeoxynucleotides are then hybridized to one or more miRNA-specific probe oligonucleotides (e.g., a microarray that comprises miRNA-specific probe oligonucleotides) to provide a hybridization profile for the test sample, and the test sample hybridization profile is compared to a hybridization profile generated from a control sample. An alteration in the signal of at least one miRNA in the test sample relative to the control sample is indicative of the subject either having, or being at risk for developing, a particular disorder with an adverse prognosis.

In some instances, it may be desirable to simultaneously determine the expression level of a plurality of different miR gene products in a sample. In other instances, it may be desirable to determine the expression level of the transcripts of all known miR genes correlated with a particular disorder. Assessing specific expression levels for hundreds of miR genes or gene products is time consuming and requires a large amount of total RNA (e.g., at least 20 μg for each Northern blot) and autoradiographic techniques that require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format (i.e., a microarray), may be constructed containing a set of oligonucleotide (e.g., oligodeoxynucleotide) probes that are specific for a set of miR genes. Using such a microarray, the expression level of multiple microRNAs in a biological sample can be determined by reverse transcribing the RNAs to generate a set of target oligodeoxynucleotides, and hybridizing them to probe the oligonucleotides on the microarray to generate a hybridization, or expression, profile. The hybridization profile of the test sample can then be compared to that of a control sample to determine which microRNAs have an altered expression level. As used herein, “probe oligonucleotide” or “probe oligodeoxynucleotide” refers to an oligonucleotide that is capable of hybridizing to a target oligonucleotide. “Target oligonucleotide” or “target oligodeoxynucleotide” refers to a molecule to be detected (e.g., via hybridization). By “miR-specific probe oligonucleotide” or “probe oligonucleotide specific for a miR” is meant a probe oligonucleotide that has a sequence selected to hybridize to a specific miR gene product, or to a reverse transcript of the specific miR gene product.

An “expression profile” or “hybridization profile” of a particular sample is essentially a fingerprint of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell. That is, normal samples may be distinguished from corresponding disorder-exhibiting samples. Within such disorder-exhibiting samples, different prognosis states (for example, good or poor long term survival prospects) may be determined. By comparing expression profiles of disorder-exhibiting samples in different states, information regarding which genes are important (including both upregulation and downregulation of genes) in each of these states is obtained.

The identification of sequences that are differentially expressed in disorder-exhibiting samples, as well as differential expression resulting in different prognostic outcomes, allows the use of this information in a number of ways. For example, a particular treatment regime may be evaluated (e.g., to determine whether a chemotherapeutic drug acts to improve the long-term prognosis in a particular subject). Similarly, diagnosis may be done or confirmed by comparing samples from a subject with known expression profiles. Furthermore, these gene expression profiles (or individual genes) allow screening of drug candidates that suppress the particular disorder expression profile or convert a poor prognosis profile to a better prognosis profile.

Alterations in the level of one or more miR gene products in cells can result in the deregulation of one or more intended targets for these miRs, which can lead to a particular disorder. Therefore, altering the level of the miR gene product (e.g., by decreasing the level of a miR that is upregulated in disorder-exhibiting cells, by increasing the level of a miR that is downregulated in disorder-exhibiting cells) may successfully treat the disorder.

Accordingly, the present invention encompasses methods of treating a disorder in a subject, wherein at least one miR gene product is deregulated (e.g., down-regulated, upregulated) in the cells of the subject. In one embodiment, the level of at least one miR gene product in a test sample is greater than the level of the corresponding miR gene product in a control or reference sample. In another embodiment, the level of at least one miR gene product in a test sample is less than the level of the corresponding miR gene product in a control sample. When the at least one isolated miR gene product is downregulated in the test sample, the method comprises administering an effective amount of the at least one isolated miR gene product, or an isolated variant or biologically-active fragment thereof, such that proliferation of the disorder-exhibiting cells in the subject is inhibited.

For example, when a miR gene product is downregulated in a cancer cell in a subject, administering an effective amount of an isolated miR gene product to the subject can inhibit proliferation of the cancer cell. The isolated miR gene product that is administered to the subject can be identical to an endogenous wild-type miR gene product that is downregulated in the cancer cell or it can be a variant or biologically-active fragment thereof.

As defined herein, a “variant” of a miR gene product refers to a miRNA that has less than 100% identity to a corresponding wild-type miR gene product and possesses one or more biological activities of the corresponding wild-type miR gene product. Examples of such biological activities include, but are not limited to, inhibition of expression of a target RNA molecule (e.g., inhibiting translation of a target RNA molecule, modulating the stability of a target RNA molecule, inhibiting processing of a target RNA molecule) and inhibition of a cellular process associated with cancer and/or a myeloproliferative disorder (e.g., cell differentiation, cell growth, cell death). These variants include species variants and variants that are the consequence of one or more mutations (e.g., a substitution, a deletion, an insertion) in a miR gene. In certain embodiments, the variant is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a corresponding wild-type miR gene product.

As defined herein, a “biologically-active fragment” of a miR gene product refers to an RNA fragment of a miR gene product that possesses one or more biological activities of a corresponding wild-type miR gene product. As described above, examples of such biological activities include, but are not limited to, inhibition of expression of a target RNA molecule and inhibition of a cellular process associated with cancer and/or a myeloproliferative disorder. In certain embodiments, the biologically-active fragment is at least about 5, 7, 10, 12, 15, or 17 nucleotides in length. In a particular embodiment, an isolated miR gene product can be administered to a subject in combination with one or more additional anti-cancer treatments. Suitable anti-cancer treatments include, but are not limited to, chemotherapy, radiation therapy and combinations thereof (e.g., chemoradiation).

When the at least one isolated miR gene product is upregulated in the cancer cells, the method comprises administering to the subject an effective amount of a compound that inhibits expression of the at least one miR gene product, such that proliferation of the disorder-exhibiting cells is inhibited. Such compounds are referred to herein as miR gene expression-inhibition compounds. Examples of suitable miR gene expression-inhibition compounds include, but are not limited to, those described herein (e.g., double-stranded RNA, antisense nucleic acids and enzymatic RNA molecules).

In a particular embodiment, a miR gene expression-inhibiting compound can be administered to a subject in combination with one or more additional anti-cancer treatments. Suitable anti-cancer treatments include, but are not limited to, chemotherapy, radiation therapy and combinations thereof (e.g., chemoradiation).

As described herein, when the at least one isolated miR gene product is upregulated in cancer cells, the method comprises administering to the subject an effective amount of at least one compound for inhibiting expression of the at least one miR gene product, such that proliferation of cancer cells is inhibited.

The terms “treat”, “treating” and “treatment”, as used herein, refer to ameliorating symptoms associated with a disease or condition, for example, cancer and/or other condition or disorder, including preventing or delaying the onset of the disease symptoms, and/or lessening the severity or frequency of symptoms of the disease, disorder or condition. The terms “subject”, “patient” and “individual” are defined herein to include animals, such as mammals, including, but not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent, or murine species. In a preferred embodiment, the animal is a human.

As used herein, an “isolated” miR gene product is one that is synthesized, or altered or removed from the natural state through human intervention. For example, a synthetic miR gene product, or a miR gene product partially or completely separated from the coexisting materials of its natural state, is considered to be “isolated.” An isolated miR gene product can exist in a substantially-purified form, or can exist in a cell into which the miR gene product has been delivered. Thus, a miR gene product that is deliberately delivered to, or expressed in, a cell is considered an “isolated” miR gene product. A miR gene product produced inside a cell from a miR precursor molecule is also considered to be an “isolated” molecule. According to the invention, the isolated miR gene products described herein can be used for the manufacture of a medicament for treating a subject (e.g., a human).

Isolated miR gene products can be obtained using a number of standard techniques. For example, the miR gene products can be chemically synthesized or recombinantly produced using methods known in the art. In one embodiment, miR gene products are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical (part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research (Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem (Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from a plasmid include, e.g., the U6 or H1 RNA pol ITT promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the miR gene products in cells (e.g., cancerous cells, cells exhibiting a myeloproliferative disorder).

The miR gene products that are expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques. The miR gene products that are expressed from recombinant plasmids can also be delivered to, and expressed directly in, cells.

The miR gene products can be expressed from a separate recombinant plasmid, or they can be expressed from the same recombinant plasmid. In one embodiment, the miR gene products are expressed as RNA precursor molecules from a single plasmid, and the precursor molecules are processed into the functional miR gene product by a suitable processing system, including, but not limited to, processing systems extant within a cancer cell.

Selection of plasmids suitable for expressing the miR gene products, methods for inserting nucleic acid sequences into the plasmid to express the gene products, and methods of delivering the recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are incorporated herein by reference. For example, in certain embodiments, a plasmid expressing the miR gene products can comprise a sequence encoding a miR precursor RNA under the control of the CMV intermediate-early promoter. As used herein, “under the control” of a promoter means that the nucleic acid sequences encoding the miR gene product are located 3′ of the promoter, so that the promoter can initiate transcription of the miR gene product coding sequences.

The miR gene products can also be expressed from recombinant viral vectors. It is contemplated that the miR gene products can be expressed from two separate recombinant viral vectors, or from the same viral vector. The RNA expressed from the recombinant viral vectors can either be isolated from cultured cell expression systems by standard techniques, or can be expressed directly in cells (e.g., cancerous cells, cells exhibiting a myeloproliferative disorder).

In other embodiments of the treatment methods of the invention, an effective amount of at least one compound that inhibits miR expression can be administered to the subject. As used herein, “inhibiting miR expression” means that the production of the precursor and/or active, mature form of miR gene product after treatment is less than the amount produced prior to treatment. One skilled in the art can readily determine whether miR expression has been inhibited in cells using, for example, the techniques for determining miR transcript level discussed herein. Inhibition can occur at the level of gene expression (i.e., by inhibiting transcription of a miR gene encoding the miR gene product) or at the level of processing (e.g., by inhibiting processing of a miR precursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miR expression is an amount sufficient to inhibit proliferation of cells in a subject suffering from cancer and/or a myeloproliferative disorder. One skilled in the art can readily determine an effective amount of a miR expression-inhibiting compound to be administered to a given subject, by taking into account factors, such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.

One skilled in the art can also readily determine an appropriate dosage regimen for administering a compound that inhibits miR expression to a given subject, as described herein. Suitable compounds for inhibiting miR gene expression include double-stranded RNA (such as short- or small-interfering RNA or “siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such as ribozymes. Each of these compounds can be targeted to a given miR gene product and interfere with the expression (e.g., by inhibiting translation, by inducing cleavage and/or degradation) of the target miR gene product.

For example, expression of a given miR gene can be inhibited by inducing RNA interference of the miR gene with an isolated double-stranded RNA (“dsRNA”) molecule which has at least 90%, for example, at least 95%, at least 98%, at least 99%, or 100%, sequence homology with at least a portion of the miR gene product. In a particular embodiment, the dsRNA molecule is a “short or small interfering RNA” or “siRNA.”

Administration of at least one miR gene product, or at least one compound for inhibiting miR expression, will inhibit the proliferation of cells (e.g., cancerous cells, cells exhibiting a myeloproliferative disorder) in a subject who has a cancer and/or a myeloproliferative disorder. As used herein, to “inhibit the proliferation of cancerous cells or cells exhibiting a myeloproliferative disorder” means to kill the cells, or permanently or temporarily arrest or slow the growth of the cells. Inhibition of cell proliferation can be inferred if the number of such cells in the subject remains constant or decreases after administration of the miR gene products or miR gene expression-inhibiting compounds. An inhibition of proliferation of cancerous cells or cells exhibiting a myeloproliferative disorder can also be inferred if the absolute number of such cells increases, but the rate of tumor growth decreases.

A miR gene product or miR gene expression-inhibiting compound can also be administered to a subject by any suitable enteral or parenteral administration route. Suitable enteral administration routes for the present methods include, e.g., oral, rectal, or intranasal delivery. Suitable parenteral administration routes include, e.g., intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. Particularly suitable administration routes are injection, infusion and direct injection into the tumor.

The miR gene products or miR gene expression-inhibition compounds can be formulated as pharmaceutical compositions, sometimes called “medicaments,” prior to administering them to a subject, according to techniques known in the art. Accordingly, the invention encompasses pharmaceutical compositions for treating cancer and/or a myeloproliferative disorder.

The present pharmaceutical compositions comprise at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising a sequence encoding the miR gene product or miR gene expression-inhibition compound) (e.g., 0.1 to 90% by weight), or a physiologically-acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier. In certain embodiments, the pharmaceutical composition of the invention additionally comprises one or more anti-cancer agents (e.g., chemotherapeutic agents). The pharmaceutical formulations of the invention can also comprise at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising a sequence encoding the miR gene product or miR gene expression-inhibition compound), which are encapsulated by liposomes and a pharmaceutically-acceptable carrier.

Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include, e.g., physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (such as, for example, calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.

For solid pharmaceutical compositions of the invention, conventional nontoxic solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, preferably 25%-75%, of the at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising sequences encoding them). A pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, preferably 1%40% by weight, of the at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising a sequence encoding the miR gene product or miR gene expression-inhibition compound) encapsulated in a liposome as described above, and a propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can further comprise one or more anti-cancer agents. In a particular embodiment, the compositions comprise at least one miR gene product or miR gene expression-inhibition compound (or at least one nucleic acid comprising a sequence encoding the miR gene product or miR gene expression-inhibition compound) and at least one chemotherapeutic agent. Chemotherapeutic agents that are suitable for the methods of the invention include, but are not limited to, DNA-alkylating agents, anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonist agents, topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acids aptamers, and molecularly-modified viral, bacterial and exotoxic agents. Examples of suitable agents for the compositions of the present invention include, but are not limited to, cytidine arabinoside, methotrexate, vincristine, etoposide (VP-16), doxombicin (adriamycin), cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin, methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine, camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab, daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine, docetaxel and FOLFOX4.

In one embodiment, the method comprises providing a test agent to a cell and measuring the level of at least one miR gene product associated with decreased expression levels in cancerous cells. An increase in the level of the miR gene product in the cell, relative to a suitable control (e.g., the level of the miR gene product in a control cell), is indicative of the test agent being an anti-cancer agent.

Suitable agents include, but are not limited to drugs (e.g., small molecules, peptides), and biological macromolecules (e.g., proteins, nucleic acids). The agent can be produced recombinantly, synthetically, or it may be isolated (i.e., purified) from a natural source. Various methods for providing such agents to a cell (e.g., transfection) are well known in the art, and several of such methods are described hereinabove. Methods for detecting the expression of at least one miR gene product (e.g., Northern blotting, in situ hybridization, RT-PCR, expression profiling) are also well known in the art. Several of these methods are also described herein.

EXAMPLES

The invention may be better understood by reference to the following non-limiting examples, which serve to illustrate but not to limit the present invention.

The data herein show that activated human mononuclear phagocytes and THP-1 cells release microvesicles that induce the survival and differentiation of freshly isolate monocytes. While not wishing to be bound by theory, the inventors herein believe that under specific inflammatory diseases, the content of the microvesicles may be altered to rapidly induce a response. The data also show that microvesicles circulate in human peripheral blood. The circulating microvesicles regulate normal cellular homeostasis, and circulate instructions to distant cells during tissue injury and inflammation.

The microvesicles may serve as biomarkers for disease etiology and systemic mediators of the innate immune response. It is thus beneficial to be able to obtain similar information through the isolation of microvesicles in the peripheral blood instead of obtaining tissue through invasive procedures. Also, understanding the normal signature of microvesicles in the peripheral blood provides a basis for understanding events during acute inflammatory events.

As shown herein, aberrant macrophage differentiation contributes to disruption in immune homeostasis. Since monocyte maturation is induced by GM-CSF or M-CSF, the inventors initiated studies to understand the mechanisms and differences between GM-CSF- and M-CSF-mediated differentiation. The commitment to differentiate in response to GM-CSF but not M-CSF was rapid and irreversible (data not shown). Continuous GM-CSF stimulation was not required for this effect as only 4 hours of treatment induced macrophage differentiation. Similar observations were obtained in PMA-treated THP1 cells used as a model of macrophage differentiation.

Thus, the inventors determined that at least one factor was secreted upon inducing differentiation that either maintained signals or activated other cells to differentiate. Therefore, monocytes or THP1 cells were exposed to GM-CSF for 4 h or PMA for 1 h, respectively, after which cells were washed and placed in minimal media without stimulus. After 24 hours, the culture supernatants were collected and added to undifferentiated monocytes or THP1 cells. Notably, supernatants from PMA-treated THP1 cells or GM-CSF-treated monocytes differentiated monocytes and THP1 cells (data not shown).

Using the Bioplex suspension array system to detect up to 27 different cytokines in the culture supernatants, the inventors failed to detect a responsible cytokine. Since the inventors differentiated the growth factor-independent THP 1 cell line with GM-CSF-stimulated monocyte supernatants, the inventors concluded that a cytokine/growth factor was not responsible for this effect. The inventors next investigated the possibility that microvesicles were secreted in the culture supernatant to mediate myeloid maturation.

As shown in FIG. 1, monocytes treated with GM-CSF for 24 hours released significant numbers of microvesicles (dark dots) in the culture supernatant compared to untreated monocytes (light dots).

Similarly, PMA-treated THP1 cells also secreted microvesicles during differentiation (data not shown). In particular, FIG. 1 shows the differentiation induced release of microvesicles from macrophages. Peripheral blood monocytes (PBM) were untreated (light) or treated with GM-CSF (dark) for 24 h. Cell-free supernatant was collected and ultracentrifuged. The vesicles were resuspended in PBS and analyzed for size on a flow cytometry. Prior to analysis, FSS and SSC parameters were adjusted using 2 μm standard beads (not shown). Shown is representative data from three different donors.

Microvesicles from PMA-treated THP1 cells were purified and added to either freshly isolated monocytes or undifferentiated THP1 cells. The microvesicles alone induced macrophage differentiation in both cell types as indicated by morphology (see FIGS. 2A-2C) and expression of surface antigens (data not shown).

The content of these microvesicles has been analyzed. The inventors detected the presence of miRNAs in the microvesicles from PMA-treated THP1 cells (data not shown).

The inventors also evaluated circulating microvesicles and miRNA in the peripheral blood of normal volunteers. Based on size, the inventors found three subpopulations of microvesicles in the circulation (FIG. 3A). Macrophage-derived microvesicles were detected using antibodies that detect mannose receptor (CD206) and MHC II (FIG. 3B). Approximately 40% of the total microvesicles (gated region) in the plasma are derived from macrophages based on expression of either CD206 or MHCII (FIG. 3C).

The inventors further determined whether miRNA are contained in the peripheral blood microvesicles. We detected expression of numerous miRNAs. The highest detected miRNAs are shown in FIG. 8 showing Table III (n=51).

Notably, miR-146 is undetectable in the peripheral blood whereas miR-155 expression was 80-fold lower than the highest expressing miRNA. Since both miR-146 and miR-155 were elevated in our IPF patient samples, but were low to undetectable in peripheral blood from normal donors, examination of circulating miRNAs may serve as a biomarker of disease.

It is now shown herein that circulating microvesicles contain miRNAs and that circulating microvesicles can provide an avenue for the miRNAs to elicit cell-to-cell communication. The microvesicles housing miRNA can also provide insight into the genetic basis of disease and can serve as predictive biomarkers.

Also, microvesicles released during macrophage differentiation can mediate maturation of immature cells. Microvesicles collected during macrophage maturation mediate the differentiation and survival of human monocytes and contain RNA. Both miRNA and processed mRNA are responsible for the maturation signals imparted on immature cells.

Example Plasma

Microvesicles are isolated from the plasma of normal healthy individuals. RNA is isolated from both the microvesicles and matched mononuclear cells and profiled for 420 known mature miRNAs by real-time PCR. Hierarchal cluster analysis of the data sets indicated significant differences in miRNA expression between peripheral blood mononuclear cells (PBMC) and plasma microvesicles.

We observed 104 and 75 miRNAs significantly expressed in the microvesicles and PBMC, respectively. Notably, 33 miRNAs were specifically expressed microvesicles compared to the PBMC. The miRNA were subjected to computational modeling to determine the biological pathways regulated by the detected miRNAs. The majority of the microRNAs expressed in the microvesicles from the blood were predicted to regulate cellular differentiation of blood cells and metabolic pathways. Interestingly, a select few microRNAs are predicted to be important modulators of immune function.

This example is the first to identify and define miRNA expression in circulating plasma microvesicles of normal subjects.

Recent evidence reveals that genetic exchange of mRNA and miRNA between cells can be accomplished through exosome-mediated transfer (PMID: 17486113). Microvesicles are small exosomes/vesicles of endocytic origin released by normal healthy or damaged cell types (PMID: 17337785, PMID: 17409393, PMID: 16791265). Microvesicles are shed from the plasma membrane into the extracellular environment to facilitate communication between cells. Despite their small size (50 nm to 1 μm) microvesicles are enriched in bioactive molecules and are suspected to contain nucleic acid and/or protein; these cell particles play a role in growth, differentiation and cancer progression (PMID: 16453000). In the peripheral blood, two-thirds of microvesicles are derived from platelets. Platelet-derived microvesicles play a role in angiogenesis and the metastatic spread of cancers such as lung cancer (PMID: 15499615). Platelet-derived microvesicles induce an immune response upon regulating gene expression in hematopoietic, endothelial, and monocytic cells (PMID: 17378242, PMID: 17127485).

Interestingly, a connection between microvesicles and miRNA has been recently made. Recently, Valadi and colleagues reported that vesicles released from human and murine mast cell lines contain over 1200 mRNA and approximately 121 miRNA molecules (PMID: 17486113) In contrast, the present invention relates to naturally occurring human plasma and blood microvesicles containing microRNA that leads to biological effects ex vivo.

FIG. 8—Table I shows that microRNAs that are important in human diseases, including cancer and non-cancer applications. The microRNA molecules associated with increase expression in disease tissue but normally with low native or undetectable expression in human plasma microvesicles (Table I, shown in FIG. 6) provides the opportunity to define changes in health and disease and may be effective biomarkers (Bold, Increase Expression Column). Similarly, normally abundant microRNAs may decrease in human plasma microvesicles to reflect the decrease observed in tissue (Bold, Decrease Expression Column).

Considerable evidence demonstrates the importance of miRNA as an inevitable cornerstone of the human genetic system. Employing the use of microvesicles to transfer genetic material would be an efficient transfer method within the human body. Microvesicular transport of miRNAs would enable communication at long distance.

Methods

Blood Collection and Microvesicle Isolation.

Peripheral blood (40 cc) was collected in EDTA tubes from 24 females and 27 males healthy non-smoking Caucasian donors following informed consent. Collection of the blood occurred either between morning and early afternoon. The median age for female donors was 29 as well as for male donors. The peripheral blood was diluted 1:1 with sterile low endotoxin PBS, layered over ficoll-hypaque (d=1.077), and centrifuged as previously described (PMID: 16931806). The mononuclear cell fraction was washed once in PBS. The microvesicles were purified from the plasma. Briefly, the vesicles were concentrated by centrifugation at 160,000×g for 1 hr at 4° C. (PMID: 10648405).

RNA Extraction.

Total RNA was isolated by Trizol (Invitrogen, Carlsbad, Calif.) extraction method. To increase the yield of small RNAs, the RNA was precipitated overnight. RNA concentration was determined and RNA integrity was a determined by capillary electrophoresis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc, Santa Clara, Calif.). For RNA isolated from mononuclear cells, only a RNA integrity number (RIN)≧9 was used. Since the intact 18s and 28s rRNA was variable in the microvesicles, the RIN was not a constraint for these samples.

miRNA Profiling by Quantitative PCR.

The expression of 420 mature human miRNAs was profiled by real-time PCR. RNA (50 ng) was converted to cDNA by priming with a mixture of looped primers to 420 known human mature miRNAs (Mega Plex kit, Applied Biosystems, Foster City, Calif.) using previously published reverse transcription conditions (PMID: 18158130). As there is no known control miRNA in microvesicles, several internal controls were examined. Primers to the internal controls, small nucleolar (sno)RNA U38B, snoRNA U43, small nuclear (sn)RNA U6 as well as 18S and 5S rRNA were included in the mix of primers.

The expression was profiled using an Applied Bio systems 7900HT real-time PCR instrument equipped with a 384 well reaction plate. Liquid-handling robots and the Zymak Twister robot were used to increase throughput and reduce error. Real-time PCR was performed using standard conditions.

Flow Cytometry.

Peripheral blood microvesicles were directly immunostained from plasma without concentration by centrifugation. To determine the cellular origin, 0.5 cc plasma was immunostained per panel of antibodies. Panel I contained antibodies recognizing CD66b-FITC (neutrophil), CD202b (Tie2)-PE (endothelial), CD206 PE-Cy5 (macrophage/dendritic), CD79a-APC (B-cell), and CD14 Pe-Cy7 (monocyte). Panel II contained antibodies to CD41a-PE-Cy5 (platelet), CCR2-APC (monocyte), CCR3-PE (dendritic cell), CCR5-PE-Cy7 (macrophage), and CD3-Alexa 610 (T-cell). Panel III contained isotype control antibodies. The samples were analyzed on BD Aria flow cytometer (BD Biosciences San Jose, Calif.). Data was expressed as percent of gated cells.

Statistical Analysis.

To reduce background noise, the miRNAs in which 80% of individual observations had a raw CT score greater than 35 were not considered during the data analysis. The internal controls (18S, 5S, snoRNA U38B, snoRNA U43, and snRNA U6) were highly variable in the plasma microvesicles as well as significantly different levels of expression in plasma microvesicles versus peripheral blood mononuclear cells (PBMC).

Thus, to reduce bias caused by using a certain miRNA as a normalization correction factor and to reduce the sample variations among RT-PCR arrays, the miRNAs were compared between plasma microvesicles and PBMC based on their relative expression to the overall miRNA expression on each array using median normalization analysis (PMID: 16854228). Controlling gender and age of the donors, linear mixed models were used to estimate the difference of specific miRNA between plasma microvesicles and PBMC. Fold-change was calculated based on the estimated mean difference.

Heat maps were generated using the miRNA that passed the filtering criteria for each tissue and miRNAs were subjected to hierarchical clustering based on their relative mean expression. miRNA expression was also ranked based on their raw CT score for plasma microvesicles and PBMC. Additional statistical analysis such as ANOVA was performed to determine miRNAs that are significant expressed between the two treatment groups

Pathway Analysis and Prediction.

Predicted miRNAs targets were determined using miRanda (microrna.sanger.ac.uk/targets/v5/). Based on the miRanda algorithm, a score is generated for each target, only scores greater than 17 were furthered analyzed using Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, Calif.). Using this software, canonical pathways were determined based on targets of the miRNAs. The dataset was examined to determine associated pathways based on gene ontology of miRNA's targets.

Results

Peripheral Blood Microvesicle Subpopulations

Initially, we examined the cellular origin of microvesicles within the peripheral blood of normal healthy individuals. Using flow cytometry, we found that the majority of the peripheral blood microvesicles are platelet-derived (FIG. 4), as previously reported (PMID: 10648405).

We also observed a second large population of microvesicles that were derived from mononuclear cell phagocyte lineage. This population was immunostained with antibodies that detected surface antigens on mononuclear phagocytes. Notably, only a small percentage of the peripheral blood microvesicles were derived from T-cells and neutrophils. We failed to detect vesicles that originated from B-cells (data not shown). Of interest, we detected a small subpopulation of microvesicles that expressed surface antigens from endothelial cells.

miRNA Expression in Plasma Microvesicles and PBMC

To test whether miRNAs are contained in the microvesicle compartment within the peripheral blood to enable communication and influence genetic changes between different tissues within the body, we performed miRNA profiling on the purified microvesicles from the plasma. We analyzed all subpopulations of microvesicles from 51 non-smoking healthy individuals comprising of 27 males and 24 females. In order to determine whether there would be differences in miRNA expression between microvesicles and PBMC, we also purified the PBMC from each donor. Real-time PCR analysis was performed to examine the expression of 420 miRNAs. The filtered data was subjected to hierarchal cluster analysis comparing the miRNA expression profile between the PBMC and plasma samples (FIG. 5A).

All but three PBMC samples clustered separately from the microvesicle samples, indicating that the miRNA expression profile between the two groups was significantly different. Based on filtering criteria to reduce background noise, we found 104 and 75 miRNAs expressed in the microvesicles and PBMC samples, respectively (FIGS. 5B and 5C).

Of these miRNAs, 71 were shared among each sample group (FIG. 5D). Notably, only two miRNAs miR-031 and 29c were expressed solely in the PBMC samples whereas four miRNAs (miR-127, -134, -485-5p, and -432) were uniquely expressed in the plasma fraction. All 104 miRNAs that are normally expressed in the plasma are shown (Table II, shown in FIG. 7).

Age and Gender Effects

We did not observe age and/or gender effects in miRNA expression from either sample group. Notably, the median age for both female and male donors was 29 years. The oldest individual was 58 years old, while the youngest was 21 years of age. Thus, we furthered stratified the data to examine differences. Examination between age-matched samples did not reveal any significant effects on miRNA expression between PBMC and microvesicles samples. While controlling gender, we also compared the upper quartile of age with the lower quartile of age, mean age for each group was 48.9±6.2 and 21.9±1.2, respectively. However, we failed to detect significant differences in miRNA expression between the samples sets based on age (data not shown).

Comparison of miRNA Expression in PBMC and Microvesicles

Shown in Table III, FIG. 8, is the top ten expressed miRNAs in the plasma microvesicles and the PBMC from all individuals. For plasma the top ten expressed miRNAs in descending order are miR-223, -484, -191, -146a, -016, -026a, -222, -024, -126, and -32. Whereas, miR-223, -150, -146b, -016, -484, -146a, -191, -026a, -019b, and -020a were highly expressed in the PBMC. The top ten expressed miRNAs in the microvesicles were detected in 100% of the individuals. However, in the PBMC samples, all but miR-150 (98% of donors) and miR-484 (89% of donors) were observed in 100% of the individuals.

We also found that six of these miRs (miR-223, miR-484, miR-191, miR-146a, miR-26a, and miR-16) are shared among the top ten in both PBMC and microvesicles. Notably, miR-223 is the most prominently expressed miR in both compartments. Based on ranking analysis for each individual donor to determine the frequency in which the specific miRNA appeared in the top ten expressed miRNA, miR-223 had a frequency of 100% in both PBMC and microvesicles. Despite expression of miR-486 being the in the top ten expressed miRNAs in the plasma microvesicles, this miRNA was found to be expressed in the top ten of only 20% of the individuals profiled. Interestingly, the highly expressed miRNAs in the plasma microvesicles were not identified as tissue-specific miRs.

We further examined the collective function of the miRs in microvesicles and PBMC with a ranking score greater than arbitrary values of >66% and >88%, respectively (natural cut-offs from the data set). Based on this criterion, we further examined the top 9 ranked miRs from the microvesicles and PBMC samples. Thus, we analyzed the combined function of miR-223, -484, -191, -146a, -016, -026a, -222, -024, and -126 found in the plasma. For PBMC, we examined the combined function of the following miRNAs, miR-223, -150, -146b, -016, -484, -146a, -191, -026a, and -019b. Using the Sanger miRBase Target version 5, we found 1578 predicted targets of the combined miRs for the plasma microvesicles (data not shown). These combined targets were subjected to computational analysis to determine the pathways that they collectively regulate. Using the Ingenuity Pathway Analysis (IPA) software, we found canonical pathways involved in metabolism and regulation of the acquired immune system were highly regulated by the expression of these shown in miRNAs (Table IV, shown in FIG. 9, top

Of the nine miRNAs examined from the PBMC fraction, we found 1857 predicted mRNA targets (data not shown). Ultimately the top five canonical pathways regulated by these miRNAs are various amino acid and lipid metabolic pathways, among others (Table IV, shown in FIG. 9, top). We also found common predicted targets from Sanger miRBase and TargetScan and determined their function (Table IV, shown in FIG. 9, bottom).

We next examined which miRNAs were differentially expressed between microvesicles and PBMC. We found 20 miRNAs had more than a three-fold increase in expression in the PBMC fraction compared to the microvesicles samples (Table V, shown in FIG. 10). In contrast, 15 miRNAs were significantly expressed in the plasma microvesicles compared to PBMC.

FIG. 11: Table VI shows the average normalized data for all miRNAs (detector name) expressed in the PBMC and the plasma with standard deviation for each.

Discussion

In these examples, the inventors now show that miRs circulate in microvesicles under normal homeostatic conditions in the peripheral blood. Here, we demonstrate 104 miRs expressed in plasma microvesicles and miR expression was significantly different from PBMC. To date, numerous studies demonstrate the ability of miRs to regulate many cellular functions. However, these studies largely imply that the miR stays within its host cell to elicit an effect (PMID: 17923084). Our data indicates that the miRNAs contained in the microvesicles may be communication signals to distant cells to regulate cellular homeostasis.

These miRNAs in the microvesicles may circulate to different tissue targets. Further examination of the highest expressed miRNAs in the plasma microvesicles, demonstrate that many of these function to regulate hematopoiesis and cellular differentiation programs (Table III, shown in FIG. 8). For instances, expression of miR-223 regulates myeloid, granulocytic and osteoclasts differentiation (PMID: 18278031, PMID: 17471500, PMID: 16325577). It also appears to have a role in hematopoietic stem cell proliferation (PMID: 18278031). Interestingly, miR-223 is loss in acute myelogenous leukemia (AML) (PMID: 18056805). In contrast, downregulation of miR-126 occurs during megakarocyte differentiation (PMID: 16549775). Notably, expression of miR-24 is regulated by TGF-β which is a potent positive and negative regulator of hematopoiesis (PMID: 16123808, PMID: 18353861). Both miR-24 and miR-16 expressed in the microvesicles regulates red cell production (PMID: 17906079, PMID: 17976518), while miR-16 also modulates lymphoid development (PMID: 16616063). Loss of miR-16 expression has been extensively examined in chronic lymphocytic leukemia (CLL) (PMID: 17327404, PMID: 17351108).

Many miRs expressed in the plasma microvesicles also regulate the progression of the cell cycle proteins (PMID: 18365017 PMID: 17914108). MiR-222 targets p27Kip1 (PMID: 17914108) while miR-24 suppresses p16 (INK4a) (PMID: 18365017). Increased expression of miR-16 results in the accumulation of cells in G0/G1 phase of the cell cycle (PMID: 16123808). In contrast, expression of miR-126 in breast cancer cells increases cellular proliferation and tumor growth but inhibits metastases (PMID: 18185580). This occurs through the regulation of vascular cell adhesion molecule-1 (VCAM1) (PMID: 18227515).

Unlike the other miRs highly expressed in the plasma microvesicles, miR-146a appears to function at a different level. While it has been suggested that miR-146a acts as a tumor suppressor and loss of this miR is associated with the development of prostate cancer (PMID: 18174313), miR-146a also modulates immune function (PMID: 16885212, PMID: 18057241). It is possible that expression of this miR in the plasma microvesicles defines immune regulatory function (Table IV, shown in FIG. 9).

Based on IPA analysis examining gene ontology of targets, the top associated networks predicted to be influenced by miR-146a expression is cellular proliferation, immune and lymphatic system development and function. In addition, this miR is predicted to regulate innate immune responses. From the analysis, we found that LPS/IL-1 and toll-like receptor signaling are among the top five canonical pathways predicted to be regulated by this mir-146a.

To date, there is no known function for miR-484 or miR-486. Similar to miR-146a, miR-484 and miR-486 appear to function as a modulator of immune responsiveness. Notably, miR-484 is the second highest expressed miR in the microvesicles fraction based on relative expression. Prediction modeling indicates that this miR has multiple functions Like many of the other miRs expressed in the microvesicles, miR-484 is predicted to regulate hematopoiesis. In particular, NK cell signaling and TL-4 signaling pathways are predicted to be targets of miR-484, while miR-486 is proposed to regulate antigen presentation. In addition, miR-486 appears to regulate cell differentiation, proliferation and growth.

While we detected 104 miRs in the plasma microvesicles, there were many that were undetectable from the total miRs profiled. Undetectable miRs in plasma microvesicles may also serve as disease biomarkers. Recently, Lawrie et al. reported that miRs were detected in the plasma of patients with B-cell lymphoma (PMID: 18318758). This study, indicated that miR-155, miR-210 and miR-21 were elevated in the plasma from these patients and miR-21 correlated with relapse. Based on this study, we detected miR-155 and miR-21 in normal individuals, but did not find miR-210. Interestingly, we found that 75% of individuals expressed miR-155 and 60% expressed miR-21 in the plasma (data not shown).

Thus, for these miRs to be used as predictive markers of disease, each individual would require a baseline prior to detection of disease. Thus, expression of miR-210 may serve as a better marker of B-cell lymphoma. Additional relationships may exist. For instance, miR-203 was undetectable in plasma microvesicles. Elevated expression of this miR is associated with bladder carcinoma and colon adenocarcinoma and may be thus used as a biomarker (PMID: 18230780, PMID: 17826655).

A converse relation may exist for plasma miRs that are normally expressed then lost with disease. For example, in acute lymphocytic leukemia (ALL), miR-223 is downregulated (PMID: 18056805). Since miR-223 is the most prominent miR expressed in the plasma microvesicles, its reduced expression may be useful as a diagnostic marker in ALL. In addition, miRs-15a/16 are lost or downregulated in chronic lymphocytic leukemia (CLL) (PMID: 18362358). While we found miR-16 was expressed in all healthy individuals that were examined, miR-15a was only expressed in 44% of the individuals profiled (data not shown).

It is of interest that we did not detect tissue specific miRNAs in the blood of normal individuals (PMID: 18025253). The majority of the microvesicles from normal individuals are derived from blood cells. We did detect a small percentage of microvesicles derived from endothelial cells. The endothelial-derived microvesicles may increase upon endothelial cell damage Likewise, the detection of tissue specific miRs and microvesicles in the peripheral blood may be a frequent event upon tissue damage. Since tumors produce microvesicles (PMID: 16283305), these may be detected in the peripheral blood.

While it has been reported that miRs are detected in the plasma (PMID: 18318758), this is the first study to characterize all known miRs from the plasma. In this study, we controlled race as a factor.

Testing the presence, absence or alterations in levels of miRs in peripheral fluids and/or blood can be useful as biomarkers to examine various diseases, to identify unique miRNA profiles, and to be a predictor of disease. The circulating miRs contained in the microvesicles have a vital function in regulating homeostasis production of blood cells as well as metabolic functions

The relevant teachings of all publications cited herein that have not explicitly been incorporated by reference, are incorporated herein by reference in their entirety. While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

While the invention has been described with reference to various and preferred embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.

The miRs of interest are listed in public databases. In certain preferred embodiments, the public database can be a central repository provided by the Sanger Institute www.http://microrna.sanger.ac.uk/sequences/ to which miR sequences are submitted for naming and nomenclature assignment, as well as placement of the sequences in a database for archiving and for online retrieval via the world wide web. Generally, the data collected on the sequences of miRs by the Sanger Institute include species, source, corresponding genomic sequences and genomic location (chromosomal coordinates), as well as full length transcription products and sequences for the mature fully processed miRNA (miRNA with a 5′ terminal phosphate group). Another database can be the GenBank database accessed through the National Center for Biotechnology Information (NCBI) website, maintained by the National Institutes of Health and the National Library of Medicine. These databases are fully incorporated herein by reference.

miR *Biogenesis miRBase byproducts that are Mature SEQ at low level, Sequence ID function unknown Accession # Mature Sequence NO hsa-let-7a* MIMAT0004481 CUAUACAAUCUACUGUCUUUC 1 hsa-let-7a-1 MIMAT0000062 UGAGGUAGUAGGUUGUAUAGUU 2 hsa-let-7a-2 MIMAT0000062 UGAGGUAGUAGGUUGUAUAGUU 3 hsa-let-7a-3 MIMAT0000062 UGAGGUAGUAGGUUGUAUAGUU 4 hsa-let-7b MIMAT0000063 UGAGGUAGUAGGUUGUGUGGUU 5 hsa-let-7b* MIMAT0004482 CUAUACAACCUACUGCCUUCCC 6 hsa-let-7c MIMAT0000064 UGAGGUAGUAGGUUGUAUGGUU 7 hsa-let-7c* MIMAT0004483 UAGAGUUACACCCUGGGAGUUA 8 hsa-let-7d MIMAT0000065 AGAGGUAGUAGGUUGCAUAGUU 9 hsa-let-7d* MIMAT0004484 CUAUACGACCUGCUGCCUUUCU 10 hsa-let-7e MIMAT0000066 UGAGGUAGGAGGUUGUAUAGUU 11 hsa-let-7e* MIMAT0004485 CUAUACGGCCUCCUAGCUUUCC 12 hsa-let-7f-1 MIMAT0000067 UGAGGUAGUAGAUUGUAUAGUU 13 hsa-let-7f-1* MIMAT0004486 CUAUACAAUCUAUUGCCUUCCC 14 hsa-let-7f-2 MIMAT0000067 UGAGGUAGUAGAUUGUAUAGUU 15 hsa-let-7f-2* MIMAT0004487 CUAUACAGUCUACUGUCUUUCC 16 hsa-let-7g MIMAT0000414 UGAGGUAGUAGUUUGUACAGUU 17 hsa-let-7g* MIMAT0004584 CUGUACAGGCCACUGCCUUGC 18 hsa-let-7i MIMAT0000415 UGAGGUAGUAGUUUGUGCUGUU 19 hsa-let-7i* MIMAT0004585 CUGCGCAAGCUACUGCCUUGCU 20 hsa-mir-009-1 MIMAT0000441 UCUUUGGUUAUCUAGCUGUAUGA 21 hsa-mir-009-1* MIMAT0000442 AUAAAGCUAGAUAACCGAAAGU 22 hsa-mir-009-2 MIMAT0000441 UCUUUGGUUAUCUAGCUGUAUGA 23 hsa-mir-009-3 MIMAT0000441 UCUUUGGUUAUCUAGCUGUAUGA 24 hsa-mir-010a MIMAT0000253 UACCCUGUAGAUCCGAAUUUGUG 25 hsa-mir-010a* MIMAT0004555 CAAAUUCGUAUCUAGGGGAAUA 26 hsa-mir-015a MIMAT0000068 UAGCAGCACAUAAUGGUUUGUG 27 hsa-mir-015b MIMAT0000417 UAGCAGCACAUCAUGGUUUACA 28 hsa-mir-015b* MIMAT0004586 CGAAUCAUUAUUUGCUGCUCUA 29 hsa-mir-016-1 MIMAT0000069 UAGCAGCACGUAAAUAUUGGCG 30 hsa-mir-016-1* MIMAT0004489 CCAGUAUUAACUGUGCUGCUGA 31 hsa-mir-016-2 MIMAT0000069 UAGCAGCACGUAAAUAUUGGCG 32 hsa-mir-016-2* MIMAT0004518 CCAAUAUUACUGUGCUGCUUUA 33 hsa-mir-017-3-p MIMAT0000071 ACUGCAGUGAAGGCACUUGUAG 34 hsa-mir-017-5-p MIMAT0000070 CAAAGUGCUUACAGUGCAGGUAG 35 hsa-mir-018a MIMAT0000072 UAAGGUGCAUCUAGUGCAGAUAG 36 hsa-mir-018a* MIMAT0002891 ACUGCCCUAAGUGCUCCUUCUGG 37 hsa-mir-019a MIMAT0000073 UGUGCAAAUCUAUGCAAAACUGA 38 hsa-mir-019b-1 MIMAT0000074 UGUGCAAAUCCAUGCAAAACUGA 39 hsa-mir-019b-1* MIMAT0004491 AGUUUUGCAGGUUUGCAUCCAGC 40 hsa-mir-019b-2 MIMAT0000074 UGUGCAAAUCCAUGCAAAACUGA 41 hsa-mir-019b-2* MIMAT0004492 AGUUUUGCAGGUUUGCAUUUCA 42 hsa-mir-020a MIMAT0000075 UAAAGUGCUUAUAGUGCAGGUAG 43 hsa-mir-020a* MIMAT0004493 ACUGCAUUAUGAGCACUUAAAG 44 hsa-mir-020b MIMAT0001413 CAAAGUGCUCAUAGUGCAGGUAG 45 hsa-mir-021 MIMAT0000076 UAGCUUAUCAGACUGAUGUUGA 46 hsa-mir-021* MIMAT0004494 CAACACCAGUCGAUGGGCUGU 47 hsa-mir-023a MIMAT0000078 AUCACAUUGCCAGGGAUUUCC 48 hsa-mir-023a* MIMAT0004496 GGGGUUCCUGGGGAUGGGAUUU 49 hsa-mir-023b MIMAT0004587 UGGGUUCCUGGCAUGCUGAUUU 50 hsa-mir-024-1 MIMAT0000080 UGGCUCAGUUCAGCAGGAACAG 51 hsa-mir-024-1* MIMAT0000079 UGCCUACUGAGCUGAUAUCAGU 52 hsa-mir-024-2 MIMAT0000080 UGGCUCAGUUCAGCAGGAACAG 53 hsa-mir-024-2* MIMAT0004497 UGCCUACUGAGCUGAAACACAG 54 hsa-mir-025 MIMAT0000081 CAUUGCACUUGUCUCGGUCUGA 55 hsa-mir-025* MIMAT0004498 AGGCGGAGACUUGGGCAAUUG 56 hsa-mir-026a-1 MIMAT0000082 UUCAAGUAAUCCAGGAUAGGCU 57 hsa-mir-026a-1* MIMAT0004499 CCUAUUCUUGGUUACUUGCACG 58 hsa-mir-026a-2 MIMAT0000082 UUCAAGUAAUCCAGGAUAGGCU 59 hsa-mir-026a-2* MIMAT0004681 CCUAUUCUUGAUUACUUGUUUC 60 hsa-mir-026b MIMAT0000083 UUCAAGUAAUUCAGGAUAGGU 61 hsa-mir-026b* MIMAT0004500 CCUGUUCUCCAUUACUUGGCUC 62 hsa-mir-027a MIMAT0000084 UUCACAGUGGCUAAGUUCCGC 63 hsa-mir-027a* MIMAT0004501 AGGGCUUAGCUGCUUGUGAGCA 64 hsa-mir-027b MIMAT0000419 UUCACAGUGGCUAAGUUCUGC 65 hsa-mir-027b* MIMAT0004588 AGAGCUUAGCUGAUUGGUGAAC 66 hsa-mir-028-3p MIMAT0004502 CACUAGAUUGUGAGCUCCUGGA 67 hsa-mir-028-5p MIMAT0000085 AAGGAGCUCACAGUCUAUUGAG 68 hsa-mir-029a MIMAT0000086 UAGCACCAUCUGAAAUCGGUUA 69 hsa-mir-029a* MIMAT0004503 ACUGAUUUCUUUUGGUGUUCAG 70 hsa-mir-029b-1 MIMAT0000100 UAGCACCAUUUGAAAUCAGUGUU 71 hsa-mir-029b-1* MIMAT0004514 GCUGGUUUCAUAUGGUGGUUUAGA 72 hsa-mir-029b-2 MIMAT0000100 UAGCACCAUUUGAAAUCAGUGUU 73 hsa-mir-029b-2* MIMAT0004515 CUGGUUUCACAUGGUGGCUUAG 74 hsa-mir-029b-3 MIMAT0000100 UAGCACCAUUUGAAAUCAGUGUU 75 hsa-mir-029c MIMAT0000681 UAGCACCAUUUGAAAUCGGUUA 76 hsa-mir-030a MIMAT0000087 UGUAAACAUCCUCGACUGGAAG 77 hsa-mir-030a* MIMAT0000088 CUUUCAGUCGGAUGUUUGCAGC 78 hsa-mir-030b MIMAT0000420 UGUAAACAUCCUACACUCAGCU 79 hsa-mir-030b* MIMAT0004589 CUGGGAGGUGGAUGUUUACUUC 80 hsa-mir-030c-1 MIMAT0000244 UGUAAACAUCCUACACUCUCAGC 81 hsa-mir-030c-2 MIMAT0000244 UGUAAACAUCCUACACUCUCAGC 82 hsa-mir-030c-2* MIMAT0004550 CUGGGAGAAGGCUGUUUACUCU 83 hsa-mir-030d MIMAT0000245 UGUAAACAUCCCCGACUGGAAG 84 hsa-mir-030d* MIMAT0004551 CUUUCAGUCAGAUGUUUGCUGC 85 hsa-mir-031 MIMAT0000089 AGGCAAGAUGCUGGCAUAGCU 86 hsa-mir-031* MIMAT0004504 UGCUAUGCCAACAUAUUGCCAU 87 hsa-mir-032 MIMAT0000090 UAUUGCACAUUACUAAGUUGCA 88 hsa-mir-032* MIMAT0004505 CAAUUUAGUGUGUGUGAUAUUU 89 hsa-mir-034a MIMAT0000255 UGGCAGUGUCUUAGCUGGUUGU 90 hsa-mir-034a* MIMAT0004557 CAAUCAGCAAGUAUACUGCCCU 91 hsa-mir-092a-1 MIMAT0000092 UAUUGCACUUGUCCCGGCCUGU 92 hsa-mir-092a-1* MIMAT0004507 AGGUUGGGAUCGGUUGCAAUGCU 93 hsa-mir-093 MIMAT0000093 CAAAGUGCUGUUCGUGCAGGUAG 94 hsa-mir-093* MIMAT0004509 ACUGCUGAGCUAGCACUUCCCG 95 hsa-mir-095 MIMAT0000094 UUCAACGGGUAUUUAUUGAGCA 96 hsa-mir-096 MIMAT0000095 UUUGGCACUAGCACAUUUUUGCU 97 hsa-mir-096* MIMAT0004510 AAUCAUGUGCAGUGCCAAUAUG 98 hsa-mir-098 MIMAT0000096 UGAGGUAGUAAGUUGUAUUGUU 99 hsa-mir-099b MIMAT0000689 CACCCGUAGAACCGACCUUGCG 100 hsa-mir-099b* MIMAT0004678 CAAGCUCGUGUCUGUGGGUCCG 101 hsa-mir-100 MIMAT0000098 AACCCGUAGAUCCGAACUUGUG 102 hsa-mir-100* MIMAT0004512 CAAGCUUGUAUCUAUAGGUAUG 103 hsa-mir-103-1 MIMAT0000101 AGCAGCAUUGUACAGGGCUAUGA 104 hsa-mir-103-2 MIMAT0000101 AGCAGCAUUGUACAGGGCUAUGA 105 hsa-mir-105-1 MIMAT0000102 UCAAAUGCUCAGACUCCUGUGGU 106 hsa-mir-105-1* MIMAT0004516 ACGGAUGUUUGAGCAUGUGCUA 107 hsa-mir-105-2 MIMAT0000102 UCAAAUGCUCAGACUCCUGUGGU 108 hsa-mir-105-2* MIMAT0004516 ACGGAUGUUUGAGCAUGUGCUA 109 hsa-mir-106a MIMAT0000103 AAAAGUGCUUACAGUGCAGGUAG 110 hsa-mir-106a* MIMAT0004517 CUGCAAUGUAAGCACUUCUUAC 111 hsa-mir-106b MIMAT0000680 UAAAGUGCUGACAGUGCAGAU 112 hsa-mir-106b* MIMAT0004672 CCGCACUGUGGGUACUUGCUGC 113 hsa-mir-107 MIMAT0000104 AGCAGCAUUGUACAGGGCUAUCA 114 hsa-mir-122 MIMAT0000421 UGGAGUGUGACAAUGGUGUUUG 115 hsa-mir-122* MIMAT0004590 AACGCCAUUAUCACACUAAAUA 116 hsa-mir-125a-3p MIMAT0004602 ACAGGUGAGGUUCUUGGGAGCC 117 hsa-mir-125a-5p MIMAT0000443 UCCCUGAGACCCUUUAACCUGUGA 118 hsa-mir-125b-1 MIMAT0000423 UCCCUGAGACCCUAACUUGUGA 119 hsa-mir-125b-1* MIMAT0004592 ACGGGUUAGGCUCUUGGGAGCU 120 hsa-mir-125b-2 MIMAT0000423 UCCCUGAGACCCUAACUUGUGA 121 hsa-mir-125b-2* MIMAT0004603 UCACAAGUCAGGCUCUUGGGAC 122 hsa-mir-126 MIMAT0000445 UCGUACCGUGAGUAAUAAUGCG 123 hsa-mir-126* MIMAT0000444 CAUUAUUACUUUUGGUACGCG 124 hsa-mir-127-3p MIMAT0000446 UCGGAUCCGUCUGAGCUUGGCU 125 hsa-mir-127-5p MIMAT0004604 CUGAAGCUCAGAGGGCUCUGAU 126 hsa-mir-128-1 MIMAT0000424 UCACAGUGAACCGGUCUCUUU 127 hsa-mir-128-2 MIMAT0000424 UCACAGUGAACCGGUCUCUUU 128 hsa-mir-130a MIMAT0000425 CAGUGCAAUGUUAAAAGGGCAU 129 hsa-mir-130a* MIMAT0004593 UUCACAUUGUGCUACUGUCUGC 130 hsa-mir-130b MIMAT0000691 CAGUGCAAUGAUGAAAGGGCAU 131 hsa-mir-130b* MIMAT0004680 ACUCUUUCCCUGUUGCACUAC 132 hsa-mir-132 MIMAT0000426 UAACAGUCUACAGCCAUGGUCG 133 hsa-mir-132* MIMAT0004594 ACCGUGGCUUUCGAUUGUUACU 134 hsa-mir-133a-1 MIMAT0000427 UUUGGUCCCCUUCAACCAGCUG 135 hsa-mir-133a-2 MIMAT0000427 UUUGGUCCCCUUCAACCAGCUG 136 hsa-mir-133b MIMAT0000770 UUUGGUCCCCUUCAACCAGCUA 137 hsa-mir-134 MIMAT0000447 UGUGACUGGUUGACCAGAGGGG 138 hsa-mir-135b MIMAT0000758 UAUGGCUUUUCAUUCCUAUGUGA 139 hsa-mir-135b* MIMAT0004698 AUGUAGGGCUAAAAGCCAUGGG 140 hsa-mir-140-3p MIMAT0004597 UACCACAGGGUAGAACCACGG 141 hsa-mir-140-5p MIMAT0000431 CAGUGGUUUUACCCUAUGGUAG 142 hsa-mir-142-3p MIMAT0000434 UGUAGUGUUUCCUACUUUAUGGA 143 hsa-mir-142-5p MIMAT0000433 CAUAAAGUAGAAAGCACUACU 144 hsa-mir-143 MIMAT0000435 UGAGAUGAAGCACUGUAGCUC 145 hsa-mir-143* MIMAT0004599 GGUGCAGUGCUGCAUCUCUGGU 146 hsa-mir-145 MIMAT0000437 GUCCAGUUUUCCCAGGAAUCCCU 147 hsa-mir-145* MIMAT0004601 GGAUUCCUGGAAAUACUGUUCU 148 hsa-mir-146a MIMAT0000449 UGAGAACUGAAUUCCAUGGGUU 149 hsa-mir-146a* MIMAT0004608 CCUCUGAAAUUCAGUUCUUCAG 150 hsa-mir-146b-3p MIMAT0004766 UGCCCUGUGGACUCAGUUCUGG 151 hsa-mir-146b-5p MIMAT0002809 UGAGAACUGAAUUCCAUAGGCU 152 hsa-mir-147 MIMAT0000251 GUGUGUGGAAAUGCUUCUGC 153 hsa-mir-148a MIMAT0000243 UCAGUGCACUACAGAACUUUGU 154 hsa-mir-148a* MIMAT0004549 AAAGUUCUGAGACACUCCGACU 155 hsa-mir-148b MIMAT0000759 UCAGUGCAUCACAGAACUUUGU 156 hsa-mir-148b* MIMAT0004699 AAGUUCUGUUAUACACUCAGGC 157 hsa-mir-149 MIMAT0000450 UCUGGCUCCGUGUCUUCACUCCC 158 hsa-mir-149* MIMAT0004609 AGGGAGGGACGGGGGCUGUGC 159 hsa-mir-150 MIMAT0000451 UCUCCCAACCCUUGUACCAGUG 160 hsa-mir-150* MIMAT0004610 CUGGUACAGGCCUGGGGGACAG 161 hsa-mir-151-3p MIMAT0000757 CUAGACUGAAGCUCCUUGAGG 162 hsa-mir-151-5p MIMAT0004697 UCGAGGAGCUCACAGUCUAGU 163 hsa-mir-155 MIMAT0000646 UUAAUGCUAAUCGUGAUAGGGGU 164 hsa-mir-155* MIMAT0004658 CUCCUACAUAUUAGCAUUAACA 165 hsa-mir-181a-1 MIMAT0000256 AACAUUCAACGCUGUCGGUGAGU 166 hsa-mir-181a-1* MIMAT0000270 ACCAUCGACCGUUGAUUGUACC 167 hsa-mir-181a-2 MIMAT0000256 AACAUUCAACGCUGUCGGUGAGU 168 hsa-mir-181a-2* MIMAT0004558 ACCACUGACCGUUGACUGUACC 169 hsa-mir-181b-1 MIMAT0000257 AACAUUCAUUGCUGUCGGUGGGU 170 hsa-mir-181b-2 MIMAT0000257 AACAUUCAUUGCUGUCGGUGGGU 171 hsa-mir-181d MIMAT0002821 AACAUUCAUUGUUGUCGGUGGGU 172 hsa-mir-182 MIMAT0000259 UUUGGCAAUGGUAGAACUCACACU 173 hsa-mir-182* MIMAT0000260 UGGUUCUAGACUUGCCAACUA 174 hsa-mir-183 MIMAT0000261 UAUGGCACUGGUAGAAUUCACU 175 hsa-mir-183* MIMAT0004560 GUGAAUUACCGAAGGGCCAUAA 176 hsa-mir-185 MIMAT0000455 UGGAGAGAAAGGCAGUUCCUGA 177 hsa-mir-185* MIMAT0004611 AGGGGCUGGCUUUCCUCUGGUC 178 hsa-mir-186 MIMAT0000456 CAAAGAAUUCUCCUUUUGGGCU 179 hsa-mir-186* MIMAT0004612 GCCCAAAGGUGAAUUUUUUGGG 180 hsa-mir-190 MIMAT0000458 UGAUAUGUUUGAUAUAUUAGGU 181 hsa-mir-191 MIMAT0000440 CAACGGAAUCCCAAAAGCAGCUG 182 hsa-mir-191* MIMAT0001618 GCUGCGCUUGGAUUUCGUCCCC 183 hsa-mir-192 MIMAT0000222 CUGACCUAUGAAUUGACAGCC 184 hsa-mir-192* MIMAT0004543 CUGCCAAUUCCAUAGGUCACAG 185 hsa-mir-193a-3p MIMAT0000459 AACUGGCCUACAAAGUCCCAGU 186 hsa-mir-193a-5p MIMAT0004614 UGGGUCUUUGCGGGCGAGAUGA 187 hsa-mir-193b MIMAT0002819 AACUGGCCCUCAAAGUCCCGCU 188 hsa-mir-193b* MIMAT0004767 CGGGGUUUUGAGGGCGAGAUGA 189 hsa-mir-195 MIMAT0000461 UAGCAGCACAGAAAUAUUGGC 190 hsa-mir-195* MIMAT0004615 CCAAUAUUGGCUGUGCUGCUCC 191 hsa-mir-196a* MIMAT0004562 CGGCAACAAGAAACUGCCUGAG 192 hsa-mir-196a-1 MIMAT0000226 UAGGUAGUUUCAUGUUGUUGGG 193 hsa-mir-196a-2 MIMAT0000226 UAGGUAGUUUCAUGUUGUUGGG 194 hsa-mir-196b MIMAT0001080 UAGGUAGUUUCCUGUUGUUGGG 195 hsa-mir-197 MIMAT0000227 UUCACCACCUUCUCCACCCAGC 196 hsa-mir-198 MIMAT0000228 GGUCCAGAGGGGAGAUAGGUUC 197 hsa-mir-199a-3p MIMAT0000232 ACAGUAGUCUGCACAUUGGUUA 198 hsa-mir-199a-5p MIMAT0000231 CCCAGUGUUCAGACUACCUGUUC 199 hsa-mir-199a-5p MIMAT0000231 CCCAGUGUUCAGACUACCUGUUC 200 hsa-mir-199b-3p MIMAT0004563 ACAGUAGUCUGCACAUUGGUUA 201 hsa-mir-199b-5p MIMAT0000263 CCCAGUGUUUAGACUAUCUGUUC 202 hsa-mir-200a MIMAT0000682 UAACACUGUCUGGUAACGAUGU 203 hsa-mir-200a* MIMAT0001620 CAUCUUACCGGACAGUGCUGGA 204 hsa-mir-200b MIMAT0000318 UAAUACUGCCUGGUAAUGAUGA 205 hsa-mir-200b* MIMAT0004571 CAUCUUACUGGGCAGCAUUGGA 206 hsa-mir-200c MIMAT0000617 UAAUACUGCCGGGUAAUGAUGGA 207 hsa-mir-200c* MIMAT0004657 CGUCUUACCCAGCAGUGUUUGG 208 hsa-mir-203 MIMAT0000264 GUGAAAUGUUUAGGACCACUAG 209 hsa-mir-204 MIMAT0000265 UUCCCUUUGUCAUCCUAUGCCU 210 hsa-mir-205 MIMAT0000266 UCCUUCAUUCCACCGGAGUCUG 211 hsa-mir-210 MIMAT0000267 CUGUGCGUGUGACAGCGGCUGA 212 hsa-mir-213 MIMAT0000256 AACAUUCAACGCUGUCGGUGAGU 213 hsa-mir-214 MIMAT0000271 ACAGCAGGCACAGACAGGCAGU 214 hsa-mir-214* MIMAT0004564 UGCCUGUCUACACUUGCUGUGC 215 hsa-mir-216a MIMAT0000273 UAAUCUCAGCUGGCAACUGUGA 216 hsa-mir-216b MIMAT0004959 AAAUCUCUGCAGGCAAAUGUGA 217 hsa-mir-217 MIMAT0000274 UACUGCAUCAGGAACUGAUUGGA 218 hsa-mir-218-1 MIMAT0000275 UUGUGCUUGAUCUAACCAUGU 219 hsa-mir-218-1* MIMAT0004565 AUGGUUCCGUCAAGCACCAUGG 220 hsa-mir-218-2 MIMAT0000275 UUGUGCUUGAUCUAACCAUGU 221 hsa-mir-218-2* MIMAT0004566 CAUGGUUCUGUCAAGCACCGCG 222 hsa-mir-221 MIMAT0000278 AGCUACAUUGUCUGCUGGGUUUC 223 hsa-mir-221* MIMAT0004568 ACCUGGCAUACAAUGUAGAUUU 224 hsa-mir-222 MIMAT0000279 AGCUACAUCUGGCUACUGGGU 225 hsa-mir-222* MIMAT0004569 CUCAGUAGCCAGUGUAGAUCCU 226 hsa-mir-223 MIMAT0000280 UGUCAGUUUGUCAAAUACCCCA 227 hsa-mir-223* MIMAT0004570 CGUGUAUUUGACAAGCUGAGUU 228 hsa-mir-224 MIMAT0000281 CAAGUCACUAGUGGUUCCGUU 229 hsa-mir-302a MIMAT0000684 UAAGUGCUUCCAUGUUUUGGUGA 230 hsa-mir-302a* MIMAT0000683 ACUUAAACGUGGAUGUACUUGCU 231 hsa-mir-302b MIMAT0000715 UAAGUGCUUCCAUGUUUUAGUAG 232 hsa-mir-302b* MIMAT0000714 ACUUUAACAUGGAAGUGCUUUC 233 hsa-mir-302c MIMAT0000717 UAAGUGCUUCCAUGUUUCAGUGG 234 hsa-mir-302c* MIMAT0000716 UUUAACAUGGGGGUACCUGCUG 235 hsa-mir-302d MIMAT0000718 UAAGUGCUUCCAUGUUUGAGUGU 236 hsa-mir-302d* MIMAT0004685 ACUUUAACAUGGAGGCACUUGC 237 hsa-mir-302e MIMAT0005931 UAAGUGCUUCCAUGCUU 238 hsa-mir-302f MIMAT0005932 UAAUUGCUUCCAUGUUU 239 hsa-mir-320a MIMAT0000510 AAAAGCUGGGUUGAGAGGGCGA 240 hsa-mir-320b-1 MIMAT0005792 AAAAGCUGGGUUGAGAGGGCAA 241 hsa-mir-320b-2 MIMAT0005792 AAAAGCUGGGUUGAGAGGGCAA 242 hsa-mir-320c-1 MIMAT0005793 AAAAGCUGGGUUGAGAGGGU 243 hsa-mir-320c-2 MIMAT0005793 AAAAGCUGGGUUGAGAGGGU 244 hsa-mir-320d-1 MIMAT0006764 AAAAGCUGGGUUGAGAGGA 245 hsa-mir-320d-2 MIMAT0006764 AAAAGCUGGGUUGAGAGGA 246 hsa-mir-324-3p MIMAT0000762 ACUGCCCCAGGUGCUGCUGG 247 hsa-mir-324-5p MIMAT0000761 CGCAUCCCCUAGGGCAUUGGUGU 248 hsa-mir-326 MIMAT0000756 CCUCUGGGCCCUUCCUCCAG 249 hsa-mir-328 MIMAT0000752 CUGGCCCUCUCUGCCCUUCCGU 250 hsa-mir-330-3p MIMAT0000751 GCAAAGCACACGGCCUGCAGAGA 251 hsa-mir-330-5p MIMAT0004693 UCUCUGGGCCUGUGUCUUAGGC 252 hsa-mir-331-3p MIMAT0000760 GCCCCUGGGCCUAUCCUAGAA 253 hsa-mir-331-5p MIMAT0004700 CUAGGUAUGGUCCCAGGGAUCC 254 hsa-mir-335 MIMAT0000765 UCAAGAGCAAUAACGAAAAAUGU 255 hsa-mir-335* MIMAT0004703 UUUUUCAUUAUUGCUCCUGACC 256 hsa-mir-339-3p MIMAT0004702 UGAGCGCCUCGACGACAGAGCCG 257 hsa-mir-339-5p MIMAT0000764 UCCCUGUCCUCCAGGAGCUCACG 258 hsa-mir-340 MIMAT0004692 UUAUAAAGCAAUGAGACUGAUU 259 hsa-mir-340* MIMAT0000750 UCCGUCUCAGUUACUUUAUAGC 260 hsa-mir-342-3p MIMAT0000753 UCUCACACAGAAAUCGCACCCGU 261 hsa-mir-342-5p MIMAT0004694 AGGGGUGCUAUCUGUGAUUGA 262 hsa-mir-345 MIMAT0000772 GCUGACUCCUAGUCCAGGGCUC 263 hsa-mir-361-3p MIMAT0004682 UCCCCCAGGUGUGAUUCUGAUUU 264 hsa-mir-361-5p MIMAT0000703 UUAUCAGAAUCUCCAGGGGUAC 265 hsa-mir-370 MIMAT0000722 GCCUGCUGGGGUGGAACCUGGU 266 hsa-mir-374a MIMAT0000727 UUAUAAUACAACCUGAUAAGUG 267 hsa-mir-374b MIMAT0004955 AUAUAAUACAACCUGCUAAGUG 268 hsa-mir-376a* MIMAT0003386 GUAGAUUCUCCUUCUAUGAGUA 269 hsa-mir-376a-1 MIMAT0000729 AUCAUAGAGGAAAAUCCACGU 270 hsa-mir-376a-2 MIMAT0000729 AUCAUAGAGGAAAAUCCACGU 271 hsa-mir-376b MIMAT0002172 AUCAUAGAGGAAAAUCCAUGUU 272 hsa-mir-376c MIMAT0000720 AACAUAGAGGAAAUUCCACGU 273 hsa-mir-378 MIMAT0000732 ACUGGACUUGGAGUCAGAAGG 274 hsa-mir-378* MIMAT0000731 CUCCUGACUCCAGGUCCUGUGU 275 hsa-mir-382 MIMAT0000737 GAAGUUGUUCGUGGUGGAUUCG 276 hsa-mir-411 MIMAT0003329 UAGUAGACCGUAUAGCGUACG 277 hsa-mir-411* MIMAT0004813 UAUGUAACACGGUCCACUAACC 278 hsa-mir-423 MIMAT0004748 UGAGGGGCAGAGAGCGAGACUUU 279 hsa-mir-423* MIMAT0001340 AGCUCGGUCUGAGGCCCCUCAGU 280 hsa-mir-425-3p MIMAT0001343 AUCGGGAAUGUCGUGUCCGCCC 281 hsa-mir-425-5p MIMAT0003393 AAUGACACGAUCACUCCCGUUGA 282 hsa-mir-432 MIMAT0002814 UCUUGGAGUAGGUCAUUGGGUGG 283 hsa-mir-432* MIMAT0002815 CUGGAUGGCUCCUCCAUGUCU 284 hsa-mir-433 MIMAT0001627 AUCAUGAUGGGCUCCUCGGUGU 285 hsa-mir-484 MIMAT0002174 UCAGGCUCAGUCCCCUCCCGAU 286 hsa-mir-485-3p MIMAT0002176 GUCAUACACGGCUCUCCUCUCU 287 hsa-mir-485-5p MIMAT0002175 AGAGGCUGGCCGUGAUGAAUUC 288 hsa-mir-486-3p MIMAT0004762 CGGGGCAGCUCAGUACAGGAU 289 hsa-mir-486-5p MIMAT0002177 UCCUGUACUGAGCUGCCCCGAG 290 hsa-mir-487a MIMAT0002178 AAUCAUACAGGGACAUCCAGUU 291 hsa-mir-487b MIMAT0003180 AAUCGUACAGGGUCAUCCACUU 292 hsa-mir-532 MIMAT0002888 CAUGCCUUGAGUGUAGGACCGU 293 hsa-mir-532-5p MIMAT0004780 CCUCCCACACCCAAGGCUUGCA 294 hsa-mir-539 MIMAT0003163 GGAGAAAUUAUCCUUGGUGUGU 295 hsa-mir-574-3p MIMAT0003239 CACGCUCAUGCACACACCCACA 296 hsa-mir-574-5p MIMAT0004795 UGAGUGUGUGUGUGUGAGUGUGU 297 hsa-mir-584 MIMAT0003249 UUAUGGUUUGCCUGGGACUGAG 298 hsa-mir-628-3p MIMAT0003297 UCUAGUAAGAGUGGCAGUCGA 299 hsa-mir-628-5p MIMAT0004809 AUGCUGACAUAUUUACUAGAGG 300 hsa-mir-643 MIMAT0003313 ACUUGUAUGCUAGCUCAGGUAG 301 hsa-mir-660 MIMAT0003338 UACCCAUUGCAUAUCGGAGUUG 302

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1. A method of diagnosing or prognosticating a colorectal cancer in a subject, comprising: i) determining a level of at least one miR gene product in microvesicles isolated from a sample from the subject, wherein the at least one miR gene product comprises miR-19a, miR-21, miR-127, miR-31, miR-96, miR-135b, miR-183, miR-30c, miR-133a, mir-143, miR-133b and miR-145; and ii) comparing the level of the at least one miR gene product in the sample to a control, wherein an increase in the level of at least one of miR-19a, miR-21, miR-127, miR-31, miR-96, miR-135b and miR-183, and/or a decrease in the level of at least one of miR-30c, miR-133a, mir-143, miR-133b and miR-145, in the sample from the subject, relative to that of the control, is diagnostic or prognostic of the colorectal cancer. 2-33. (canceled)
 34. The method of claim 1, wherein the control is selected from the group consisting of: i) a reference standard; ii) the level of the at least one miR gene product from a subject that does not have colorectal cancer; and iii) the level of the at least one miR gene product from a sample of the subject that does not exhibit colorectal cancer.
 35. The method of claim 1, wherein the subject is a human. 36-44. (canceled)
 45. The method of claim 1, wherein determining the level of at least one miR gene product comprises: (a) labeling the miR isolated from the sample from the subject; (b) hybridizing the miR to an miR array; and (c) determining miR hybridization to the array.
 46. The method of claim 1, wherein identifying miR differentially expressed comprises generating an miR profile for the sample and evaluating the miR profile to determine whether miR in the sample are differentially expressed compared to a normal sample. 47-50. (canceled)
 51. The method of claim 1, wherein the sample comprises a peripheral fluid.
 52. The method of claim 51, wherein the peripheral fluid comprises blood or a fraction thereof.
 53. The method of claim 1, wherein determining the level of at least one miR gene product comprises using real-time PCR.
 54. The method of claim 1, wherein the microvesicles are isolated from the sample using ultracentrifugation and/or flow cytometry. 